LOCUS       sequence-435531-        8274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In mammalian cells expresses a Quartet of receptor-binding domains 
            (RBDs) from the sarbecoviruses SHC014, Rs4081, RaTG13, and 
            SARS-CoV-2 each separated by GS-linkers with a C-terminal SpyTag 
            fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8274)
  AUTHORS   Hills RA, Tan TK, Cohen AA, Keeffe JR, Keeble AH, Gnanapragasam PNP,
            Storm KN, Rorick AV, West AP Jr, Hill ML, Liu S, Gilbert-Jaramillo 
            J, Afzal M, Napier A, Admans G, James WS, Bjorkman PJ, Townsend AR, 
            Howarth MR
  TITLE     Proactive vaccination using multiviral Quartet Nanocages to elicit 
            broad anti-coronavirus responses.
  JOURNAL   Nat Nanotechnol. 2024 May 6. doi: 10.1038/s41565-024-01655-9.
  PUBMED    38710880
REFERENCE   2  (bases 1 to 8274)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Nanotechnol. 2024 May 6. doi: 10.1038/s41565-024-01655-9."
COMMENT     Sequence Label: Quartet-SpyTag
FEATURES             Location/Qualifiers
     source          1..8274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1010..1045
                     /label=SnoopTag2
     CDS             complement(3408..3425)
                     /codon_start=1
                     /product="tetracysteine peptide that binds biarsenical 
                     labeling reagents"
                     /label=tetracysteine tag
                     /translation="CCPGCC"
     CDS             3701..3769
                     /codon_start=1
                     /product="peptide derived from the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogTag
                     /note="forms a covalent bond with the DogCatcher protein 
                     when inserted into an intra-domain protein loop (Keeble et 
                     al., 2022)"
                     /translation="DIPATYEFTDGKHYITNEPLPPK"
     CDS             3788..3826
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     primer_bind     complement(3898..3915)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3904..4128
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4174..4602
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4261..4280)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4471..4492
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4611..4631)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     4825..4844
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4979..5773
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5033..5052)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5643..5662
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5947..6068
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5984..6003)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6038..6057
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6117..6133)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6117..6133)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6130..6152)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6141..6157
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(6165..6171,6172..6189,6190..6195))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6210..6231
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6348..6365)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6519..7107)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6599..6618)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7278..8069,8070..8138))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7901..7920
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8139..8243)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgccaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaacgg gccctctaga ctcgagcggc cgccaccatg aacacccaga tcctggtgtt
      961 cgccctgatc gccatcattc ccaccaacgc cgacaagatc ggatctggaa aactgggcta
     1021 tattgaattt tataaagtgg aaaaaggcag tggtgagtcg ggatccggaa gagtggcccc
     1081 tagcaaagaa gtcgtgcggt tccccaatat caccaatctg tgccccttcg gcgaggtgtt
     1141 caacgccacc acatttccct ctgtgtacgc ctgggagaga aagcggatca gcaactgcgt
     1201 ggccgactac agcgtgctgt acaacagcac ctcctttagc accttcaagt gctacggcgt
     1261 gtccgccacc aagctgaacg atctgtgctt ctccaacgtg tacgccgaca gcttcgtggt
     1321 caagggcgac gatgtgcggc agattgctcc tggacagaca ggcgtgatcg ccgattacaa
     1381 ctacaagctg cctgacgact tcctgggctg tgtgctggcc tggaacacca atagcaagga
     1441 cagctccacc agcggcaact acaactacct gtacagatgg gtccgacgca gcaagctgaa
     1501 tccctacgag agggacctga gcaacgacat ctatagccct ggcggccaga gctgttctgc
     1561 cgtgggaccc aattgctaca accctctgag gccctacggc ttctttacca cagctggcgt
     1621 gggccaccag ccttacagag tggtggtgct gtcattcgag ctgctgaatg cccctgccac
     1681 agtgtgtggc cctaagctgt ctaccgacct gatcaagaac caggggagtg gtgggtccgg
     1741 gggttcggga cgggtgtccc ctacacacga ggtcgtgcgg ttccccaata tcaccaacag
     1801 atgccccttc gacaaggtgt tcaacgccag cagattcccc aacgtgtacg cctgggagcg
     1861 caccaagatc agcgattgcg tggccgatta caccgtgctg tacaactcca ccagctttag
     1921 cacctttaag tgctacggcg tgtccccaag caagctgatc gacctgtgct tcacctctgt
     1981 gtacgccgac accttcctga tccggtctag cgaagtgcga caagtggccc ctggcgaaac
     2041 aggcgtgatc gccgactaca actacaagct gcccgacgac ttcaccggct gtgtgatcgc
     2101 ctggaatacc gccaagcagg accagggcca gtactactac agaagcagca gaaagaccaa
     2161 gctcaagccc ttcgagcggg acctgacctc cgatgagaat ggcgtcagaa ccctgtccac
     2221 ctacgatttc taccccaatg tgcccatcga gtaccaggcc accagagtgg tggtgctgtc
     2281 tttcgaactg ctgaacgccc ctgccacagt gtgtggccct aaactgtcta cagccctggt
     2341 caagaaccag tgcgtgaact tcggcagcgg cggaacggga ggtagtggcc gggtgcagcc
     2401 caccgatagc atcgtgcggt tccccaatat caccaatctg tgccccttcg gcgaggtgtt
     2461 caatgccacc acattcgcct ctgtgtacgc ctggaaccgg aagcggatca gcaattgcgt
     2521 ggccgactac tccgtgctgt acaacagcac cagcttcagc accttcaagt gctacggcgt
     2581 gtcccctacc aagctgaacg acctgtgctt cacaaacgtg tacgccgaca gcttcgtgat
     2641 caccggggat gaagtgcggc agattgcccc tggacagaca ggcaagatcg ccgactacaa
     2701 ctacaagctg cccgacgact tcaccggctg tgtgattgcc tggaactcca agcacatcga
     2761 cgccaaagaa ggcgggaact tcaactacct gtaccggctg ttccggaagg ccaacctgaa
     2821 gcctttcgag cgggatatct ccaccgagat ctaccaggcc ggcagcaagc cttgtaatgg
     2881 ccagacaggc ctgaactgct actacccact gtacagatac ggcttctacc ccacagacgg
     2941 cgtgggccac cagccttata gagtggtggt gctgagcttc gagctgctga atgcccctgc
     3001 cacagtgtgc ggccctaaga aaagcaccaa tctcgtgaag aacaaatgcg tgaacttcgg
     3061 tacgggtggc agtggaggga gcgggaacat caccaatctg tgccccttcg gcgaggtgtt
     3121 caatgccaca agatttgcca gcgtgtacgc ctggaaccgg aagagaatca gcaactgcgt
     3181 ggccgactac agcgtgctgt acaatagcgc cagcttcagc accttcaagt gctacggcgt
     3241 gtcccctacc aagctgaacg acctgtgctt caccaatgtg tacgccgaca gcttcgtgat
     3301 cagaggcgac gaagttcggc agatcgctcc tggacagaca ggcaagatcg ccgattacaa
     3361 ctacaagctg cccgacgact tcaccggctg cgtgatcgcc tggaatagca acaacctgga
     3421 cagcaaagtc ggcggcaact acaactacct gtaccggctg ttccggaagt ccaacctgaa
     3481 gcctttcgag cgggacatca gcaccgagat ctatcaggcc ggcagcaccc cttgtaatgg
     3541 cgtggaaggc ttcaactgct acttcccact gcagtcctac ggcttccagc ctacaaatgg
     3601 cgtcggctac cagccttaca gagtggtggt gctgtccttc gaactgctgc atgcccctgc
     3661 taccgtgtgc ggccctaaaa aaaccggagg aggtggcggt gatattccgg ctacatacga
     3721 attcaccgat ggtaaacatt atatcaccaa tgaaccgctg ccgccgaaag gtggtggcgg
     3781 aggcggagcc cacatcgtga tggttgatgc ctacaagccc accaaatagt gaattccacc
     3841 acactggact agtggatccg agctcggtac caagcttaag tttaaaccgc tgatcagcct
     3901 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3961 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4021 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     4081 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     4141 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     4201 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4261 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4321 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4381 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4441 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4501 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     4561 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
     4621 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4681 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4741 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4801 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     4861 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     4921 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     4981 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     5041 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     5101 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     5161 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     5221 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     5281 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     5341 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     5401 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     5461 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     5521 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     5581 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     5641 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     5701 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     5761 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     5821 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5881 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5941 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     6001 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     6061 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     6121 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     6181 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     6241 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6301 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     6361 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     6421 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     6481 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     6541 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     6601 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     6661 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6721 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6781 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6841 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6901 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6961 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     7021 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     7081 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     7141 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     7201 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     7261 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     7321 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     7381 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     7441 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     7501 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     7561 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     7621 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     7681 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7741 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7801 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7861 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7921 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7981 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     8041 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     8101 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     8161 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     8221 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
